Rapamycin and process of preparation

ABSTRACT

Antibiotic rapamycin is producible by culturing Streptomyces hygroscopicus NRRL 5491 in an aqueous nutrient medium. Rapamycin has antifungal properties. Methods for its preparation and use are disclosed.

United States Patent 1 Sehgal et al.

[ Dec. 30, 1975 [73] Assignee: Ayerst McKenna and Harrison Ltd.,Montreal, Canada [22] Filed: Apr. 12, 1974 [21] Appl. No.: 460,665

Related US. Application Data [63] Continuation-impart of Ser. No.293,699, Sept. 29,

1972, abandoned.

52 us. Cl. 424 122; 195/80 R [51] int. 01. l. A61K 35/00 [58] Field ofSearch 424/122; 195/80 [56] References Cited OTHER PUBLICATIONS Miller,The Pfizer Handbook of Microbial Metabolites McGraw-Hill Book Co. l nc.,N.Y., N.Y., 1961, p. 580.

Primary Examiner-Jerome D. Goldberg [57] ABSTRACT Antibiotic rapamycinis producible by culturing Streptomyces hygroscopicus NRRL 5491 in anaqueous nutrient medium. Rapamycin has antifungal properties. Methodsfor its preparation and use are disclosed.

4 Claims, 2 Drawing Figures US. Patent Dec. 30, 1975 Sheet 1 of23,929,992

CON

00v 000 Dow i=2 $252 w a coooofi 00: 002 002 ooou comm ooom comm OOOQIII!

Om Om 2 o w R o m w m $20525 EOZm w aauv'auos'av US. Patent Dec. 30,1975 Sheet 2 of2 3,929,992

5 2k O Q- 0 QM O Qm 0 0 OK 06 1- d L m W w 6 0 06 OK 0 0 Qm o6 QM od 32.:

RAPAMYCIN AND PROCESS OF PREPARATION This application is acontinuation-in-part of our earlier application Ser. No. 293,699, filedSept. 29, 1972 now abandoned.

BACKGROUND OF THE INVENTION a. Field of Invention This invention relatesto an antibiotic, a new composition of matter calling rapamycin, and toa process for its preparation.

b. Description of Prior Art The antibiotic of this invention is readilydistinguished from prior art compounds of its class by its profoundantifungal activity and its relatively low order of toxicity.

More explicitly, the ultra violet spectrum of rapamycin, noted herein,indicates that this compound belongs to the class of antibiotics knownas triene antibiotics. In this particular class there are only fivecompounds reported previously. Trienine, A. Aszalos et al., J.Antibiotics, 2 l 61 l (1968) is a triene antibiotic with antitumoractivity which also shows marked activity against gram positiveorganisms and only marginal activity against Candida strains. Theantifungal triene reported by J. J. Armstrong, et al., Nature, 206, 399(1965) and Mycotrienin reported by C. Coronelli et al., J. Antibiotics,20, 329 (1967) are probably identical. Both have low antifungal activity(MIC against C. albicans: 5 pig/ml) and high toxicity (LD in mice:mg/kg). The remaining two antibiotics Resistaphylin, S. Aezaiva et al.,.I. Antibiotics, 24, 393 (1971) and Proticin, G. Nesemann et al.,Naturwissenschaften, 59, 81 (1972)- are readily distinguished from thecompound of the present invention in that they exhibit antibacterialwithout any antifungal activity.

BRIEF SUMMARY OF THE INVENTION Rapamycin is a chemical compoundproducible by culturing a rapamycin-producing organism in an aqueousnutrient medium. The compound has the property of adversely affectingthe growth of fungi, for example, Candida albicans and Microsporumgypseum. Accordingly, rapamycin may be used to prevent the growth of orreduce the number of certain fungi in various environments.

The rapamycin producing organism used for this invention, Streptomyceshygroscopicus NRRL 5491, was obtained from Easter Island soils andsamples thereof have been deposited without restrictions with theNorthern Utilization and Research Division, Agricultural ResearchService, US Department of Agriculture, Peoria, 111., U.S.A.

It is to be understood that the invention is not limited to the use ofthe particular organism herein described, but includes variations andmutants obtained by natural selection or by treatment of themicroorganism with, for instance, ultraviolet rays, X-rays, N-methyl-N'-nitro-N-nitroso-guanidine, manganese chloride, camphor, nitrogenmustards, and the like,'as well as polyploids of the various mutants.

Streptomyces hygroscopicus NRRL 5491 develops abundantly in culturemedia usually employed for cultivation of other organisms of the samegenus. It is capable of growing at temperatures ranging from to 35C.,preferably at about 28C, on Czapeks agar, glucose asparagine agar,glycerol asparagine agar,

starch agar and peptone beef agar. Also, the organism grows very well onyeast extract agar, malt extract agar, starch-inorganic salts agar,oatmeal agar, oatmealtomato agar and Bennets agar. On potato slicesthere is no aerial mycelium, but substrate growth is well developed andbuff in color. On all media, the aerial growth is at first white thengrayish with black spots. Sporophores areoften compact, forming a spiralof more than ten spores. Substrate growth is light yellow to almostcolorless and in some media pale brown. Occasionally a yellowish pigmentis produced. The organism is H S- and melamine-negative.

Carbohydrate utilization by Streptamyces hygroscopicus NRRL 5491 wasstudied in carbon utilization agar ([8? Medium 9) according to theprocedure standardized by the International Streptomyces Project (ISP).

The best utilized carbohydrates were D-glucose, inositol, D-fructose andD-mannitol; less well utilized carbohydrates were rhamnose, raffinose,xylose, starch and arabinose. Carbohydrates not utilized were sucroseand cellulose.

The environment and nutritional requirements for the fermentation ofStreptomyces hygroscopicus NRRL 5491 are similar to those necessary forthe production of antibiotics by other aerobic microorganisms. Thus,aerobiosis can be sustained in a liquid nutrient medium inoculated witha sterile culture incubated in flasks placed on shaking machines. Forindustrial production, metal tanks with internal aeration and agitationby means of paddles can be substituted. Rapamycin is also produced bysurface cultivation. The microorganism requires as nutrient elementsassimilable carbon and organic nitrogenous substances. The presence ofmineral salts is desirable. Cultivation is best effected when theinitial pH of the culture medium is between 6.5 and 7.5, the optimum pHbeing around 6.8-7.3.

The utilizable sources of assimilable carbon for the production of theantibiotic are very diverse, there being included sugars (for example,glucose, D-fructose, D-mannitol, maltose, arabinose, rhamnose,raffinose, xylose, and the like), dextrin, starches of different typesof origin, glycerol (and other polyalcohols), inositol and animal andvegetable fats, as well as esters thereof. The sources of organicassimilable nitrogen which actively stimulate growth and favorproduction of rapamycin are substances such as soybean meal, cotton mealand other vegetable meals (whole or partially or totally defatted), meatflours or animal viscera, various peptones, casein hydrolysates, soybeanhydrolysates, yeast hydrolysates, lactalbumin, wheat glutins, distillerssolubles, corn steeps, molasses, urea and amino acids.

Mineral salts, such as the chlorides, nitrates, sulfates, carbonates andphosphates of sodium, potassium, ammonium and calcium, should beincluded in appropriate concentrations. The nutritive medium shouldcontain a number of trace elements such as magnesium, iron, manganese,and zinc.

The inoculum of the above medium for the fermentation is provided with afresh slant of Streptomyces hygroscopicus NRRL 5491.

Under the described conditions and with the temperature of cultivationat about 20-35C, preferably at about 25C, maximum production ofrapamycin in tanks is obtained in from about 2 to about 8 days.Alternatively, the pH may be controlled during fermentation in tanks andmaintained at about pH 6.0, and glu- 3 cose may be added continuouslyfrom about 2 days after beginning to the end of fermentation, thusobtaining maximum yields in about 4 to 5 days.

Thereafter, a variety of procedures may be employed in the isolation andpurification of rapamycin, for example, solvent extraction, partitionchromatography, silica gel chromatography, liquid-liquid distribution ina Craig apparatus, and crystallization from solvents. Solvent extractionprocedures are preferred for commercial recovery inasmuch as they areless time consuming and less expensive.

Generally speaking, rapamycin may be harvested by one of the followingmethods.

a. The fermentation mixture is extracted with a substantiallywater-immiscible solvent, preferably a lower alkanol, for examplen-butanol, n-pentanol or the commercial mixture of pentanols known asPentasol or n-hexanol, or a substantially water-immiscible lower alkyllower alkanoate, for example, ethyl acetate, butyl acetate, amyl acetateor the commercially available mixture of amyl acetates, or asubstantially waterimmiscible halogenated aliphatic hydrocarbomforexample, chloroform, methylene dichloride or dichloroethane. Theextracts are dried and concentrated under reduced pressure to yield anoily residue which is in turn extracted with a water-miscible solvent,preferably a lower alkanol, for example methanol or ethanol. Saidlast-named extracts are filtered through diatomaceous earth (Celite),and the filtrate concentrated under reduced pressure to yield an oilyresidue containing crude rapamycin.

b. The fermentation mixture is filtered through a pad of diatomaceousearth (Celite) and the filter cake containing the mycelium is extractedas described below under (c). The filtrate, i.e. the mycelium-freefermentation mixture, is extracted several times with a substantiallywater-immiscible solvent, for example, a lower alkanol, lower alkyllower alkanoate or halogenated aliphatic hydrocarbon as exemplifiedabove in section (a). The extracts are dried and concentrated underreduced pressure to yield an oily residue which is extracted with awater-miscible solvent, preferably a lower alkanol, for example methanolor ethanol. Said last-named extracts are treated in the same manner asdescribed above under (a) to yield an oily residue containing cruderapamycin.

c. The mycelium is separated from the fermentation mixture and extractedwith a suitable water-miscible solvent, preferably a lower alkanol, forexample methanol or ethanol. The extract is concentrated by evaporationto the aqueous phase, which in turn is extracted with a substantiallywater-immiscible solvent, such as a lower alkyl lower alkanoate,halogenated aliphatic hydrocarbon or a substantially water-immisciblelower alkanol as described above or an aromatic hydrocarbon, for examplebenzene or toluene. The latter extract is evaporated under reducedpressure to yield an oily residue containing crude rapamycin.

The crude rapamycin obtained by any of the processes described insections (a), (b) or (c) is then purified by a variety of methods, forexample, see above. Preferred methods include absorption of the cruderapamycin on an absorbent, for instance charcoal or silica gel from asolution in a substantially non-polar, first solvent, followed byelution therefrom with a second solvent, more polar than said firstsolvent.

,DETAILS OF THE INVENTION Rapamycin is useful as an antifungal agentagainst a number of pathogenic fungi; for example, Candida albicans, andother Candida species, Micmsporum gypseum, Trichophyton mentagrophytes,Aspergillus sp., and Sporotrichum sp..

The inhibitory activity of rapamycin is especially pronounced againstCandida albicans and said last organism may be used advantageously forassay purposes.

The antifungal activity of this compound is demonstrable in standardtests used for this purpose, for example, in the tests described inAntiseptics, Disinfectants, Fungicides and Sterilization, G. F. Reddish,Ed., 2nd ed., Lea and Febiger, Philadelphia, 1957 or by D. C. Grove andW. A. Randall in Assay Methods of Antibiotics, Med. Encycl. lnc., NewYork 1955.

When the antibiotic of this invention is employed as an antifungal agentin warm-blooded animals, e.g. rats, it may be used alone or incombination with pharmaceutically acceptable carriers, the proportion ofwhich is determined by the solubility and chemical nature of thecompound, chosen route of administration and standard biologicalpractice. For example, an antifungally effective amount of theantibiotic may be administered orally in solid form containing suchexcipients as starch, sugar, certain types of clay and so forth.Similarly, such an amount may also be administered orally in the form ofsolutions or suspensions, or the antibiotic :may be injectedparenterally. For parenteral administration the antibiotic may be usedin the form of a sterile solution or suspension containing other solutesor suspending agents, for example, enough saline or glucose to make thesolution isotonic, bile salts, acacia, gelatin, sorbitan monoleate,polysorbate (oleate esters of sorbitol and its anhydrides copolymerizedwith ethylene oxide) and the like.

The dosage of the present antibiotic will vary with the form ofadministration and the particular compound chosen. Furthermore, it willvary with the particular host under treatment. Generally, treatment isinitiated with small dosages substantially less than the optimum dose ofthe compound. Thereafter, the dosage is increased by small incrementsuntil the optimum effect under the circumstances is reached. in general,the compound of this invention is most desirably administered at aconcentration level that will generally afford antifungally effectiveresults without causing any harmful or deleterious side effects andpreferably at a level that is in a range of from about 1.0 mg to about250 mg per kilo per day, although as aforementioned variations willoccur. However, a dosage level that is in the range of from about 10 mgto about mg per kilo per day is most desirably employed in order toachieve effective results.

. In addition, the agent may be employed topically. For topicalapplication it may-be formulated in the form of solutions, creams, orlotions in pharmaceutically acceptable vehicles containing 0.1-5 percent, preferably 2 per cent of the agent, and may be administeredtopically to the infected area of the skin.

Rapamycin may also be used for cleaning and disinfecting laboratoryequipment, surgical instruments, locker rooms, or shower rooms ofsensitive fungus organisms. For such purposes it is preferred to use01-10% solutions of rapamycin in a lower alkanol, preferably methanol,diluted with 10-100 volumes of water containing 0001-0. 1 ofa non-ionicsurface-active agent, for example, polysorbate 80 U.S.P., immediatelybefore applying it to the objects to be cleaned and disinfected.

PREPARATION In one embodiment of this invention rapamycin is prepared inthe followingmanner: 4

A suitable fermenter is charged with production meis reached in thefermentation mixture after 2-8 days,

usually after about 5 days, as determined by the cup plate method andCandida albicans as the test organism. The mycelium is harvested byfiltration with diatomaceous earth. Rapamycin is then extracted from themycelium with a water-miscible solvent, for example a lower alkanol,preferably methanol or ethanol. The latter extract is then concentrated,preferably under reduced pressure, and the resulting aqueous phase isextracted with a water-immiscible solvent. A preferred water-immisciblesolvent for this purpose is methylene dichloride although chloroform,carbon tetrachloride, benzene, n-butanol and the like may also be used.The latter extract is concentrated, preferably under reduced pressure,to afford the crude product as an oil.

The product may be purified further by a variety of methods. Among thepreferred methods of purification is to dissolve the crude product in asubstantially nonpolar, first solvent, for example petroleum ether orhexane, and to treat the resulting solution with a suit able absorbent,for example charcoal or silica gel, so that the antibiotic becomesabsorbed on the absorbant. The absorbant is then separated and washed oreluted with a second solvent more polar than the first solvent, forexample ethyl acetate, methylene dichloride, or a mixture of methylenedichloride and ether (preferred). Thereafter, concentration of the washsolution or eluate affords substantially pure rapamycin. Furtherpurification is obtained by partial precipitation with a nonpolarsolvent, for example, petroleum ether, hexane, pentane and the like,from a solution of the rapamycin in a more polar solvent, for example,ether, ethyl acetate, benzene and the like. Still-further purificationis obtained by column chromatography, preferably employing silica gel,and by crystallization of the rapamycin from ether.

In another preferred embodiment of this invention a first stage inoculumof S treptomyces hygroscopicus NRRL 5491 is prepared in small batches ina medium containing soybean flour, glucose, ammonium sulfate, andcalcium carbonate incubated at about 25C at pH 7.l-7.3 for 24 hrs. withagitation, preferably on a gyrotary shaker. The growth thus obtained isused to inoculate a number of somewhat larger batches of the same mediumas described above which are incubated at about 25C and pH 7.1-7.3 for18 hrs. with agitation, preferably on a reciprocating'shaker, to obtaina sec- "ond stagc inoculum which is used to inoculate the productionstage fermenters.

6 5.86'.2.-The fermenters are inoculated with the second stage inoculumdescribed above and incubated at about 25C with' agitationand aerationwhile controlling and 'mai'ntaining the mixture at approximately pH 6.0by

addition offa base, for example, sodium hydroxide, potassium hydroxideor preferably ammonium hydroxide, as required from time to time.Addition of a source -of assimilable carbon, preferably glucose, isstarted when theconcentrationof the latter in the broth has dropped toabout 0.5% wt/vol, normally about 48 hrs after. the start offermentation, and is maintained until the end ofthe particular run. Inthis manner a fermentation broth containing about 60 ug/ml of rapamycinas determined by the assay method described above is obtained in 45days, when fermentation is stopped.

' Filtration of the'mycelium, mixing the latter with a watef-miscible'lower' alkanol, preferably methanol, followed by extraction with ahalogenated aliphatic hydrocarbon, preferably trichloroethane, andevaporation of the solvents yields a first oily residue. This first oilyresidue is dissolved in a lower aliphatic ketone, preferably acetone,filtered from insoluble impurities, the filtrate evaporated to yield asecond oily residue which is extractedjwith a water-miscible loweralkanol,

preferably methanol, and the latter extract is evaporated to yield cruderapamycin as a third oily residue. This third oily residue is dissolvedin a mixture of a lower aliphatic ketone and a lower aliphatichydrocarbon, preferably acetone-hexane, an absorbent such as charcoal orpreferably silica gel is added to adsorb the rapamycin, the latter iseluted from the adsorbate with a similar but more polar solvent mixture,for example a mixture as above but containing a higher proportion of thealiphatic ketone, the eluates are evaporated and the residue iscrystallized from diethyl ether, to yield pure crystalline rapamycin. Inthis manner a total of 45-5 8% of the rapamycin initially present in thefermentation mixture is recovered as pure crystalline rapamycin.

CHARACTERIZATION solvent systems; for example, ether-hexane 40:60 (Rf0.42), 'isopropyl alcoholvbenzene 15:85 (Rf= 0.5) and ethanol-benzene20:80 (Rf f 0.43);

d. rapamycin obtained from four successive fermentation batchesgave thefollowing values on repeated The production stage fermenters areequipped with 7 devices for controlling and maintaining pH at apredetermined level and for continuous metered addition of elementalanalyses:

AVER- e. rapamycin exhibits the following characteristic absorptionmaxima in its ultraviolet absorption spectrum ethanol):

f. the infrared absorption spectrum of rapamycin in chloroform isreproduced in FIG. 1 and shows characteristic absorption bands at 3560,3430, 1730, 1705 and 1630-1610 cm;

Further infrared absorption bands are characterized by the followingdata given in reciprocal centimeters with (s) denoting a strong, (m)denoting a medium, and (w) denoting a weak intensity band. Thisclassification is arbitrarily selected in such a manner that a band isdenoted as strong (s) if its peak absorption is more than two-thirds ofthe background in the same region; medium (m) if its peak is betweenone-third and twothirds of the background in the same region; and weak(w) if its peak is less than one-third of the background in the sameregion.

2990 cm (m) 1158 cm" (m) 2955 cm (s) 1129 cm (s) 2919 cm (s) 1080 cm (s)2858 cm (s) 1060 cm (s) 2815 cm (m) 1040 cm (m) 1440 cm (s) 1020 crn'(m) 1365 cm (m) 978 cm" (s) 1316 cm (in) 905 cm (m) 1272 cm (m) 888 cm"(w) 1178 cm (s) 866 cm- (w) g. the nuclear magnetic resonance spectrumof rapamycinin deuterochloroform is reproduced in FIG. 2;

h. the minimum inhibitory concentration of rapamycin against variousmicroorganism is listed below:

i. rapamyein exhibits a LD (i.p., mice) of 597.3 1*: 28.1 mg/kg and a LD(p.o., mice) of 2,500 mg/kg.

In protection studies, mice were infected by intravenous injection of C.albicans ATCC 11651. At 1,4 and 24 hours after infection, mice wereadministered mg/kg (s.c.) of rapamycin. At this dose 50% of the micewere protected. Treatment with 25 mg/kg (s.c.) offered completeprotection. When rapamyein was administered orally, at 10 mg/kg 4 out of10 mice survived, and at 25 mg/kg complete protection was observed.

A 1% suspension (0.2 ml) of rapamycin in water containing 1.5%polysorbate 80 (Tween 80), when injected intradermally into a rabbitsear caused no irritation. Similarly, two drops of a 0.5% suspensionapplied to a rabbit's eye caused no irritation.

The following Examples illustrate further this inventron.

EXAMPLE 1 Microorganism Streptomyces hygroscopicus NRRL 5491 was grownand maintained on oatmeal-tomato paste agar slants (T. G. Pridham, etal., Antibiotic Annual 1956-1957, Medical Encyclopedia Inc., New York,p. 947) and in Roux bottles containing the same medium. Good growth wasobtained after 7 days of incubation at 28C. Spores from one Roux bottlewere washed off and suspended into 50 ml of sterile distilled water.This suspension was used to inoculate the first stage inoculum.

The first-stage inoculum medium consisted of Emerson broth [R. L.Emerson et al., J. Bacteriol, 52, 357 (1946)] 0.4%; peptone, 0.4%;sodium chloride, 0.25%; yeast extract, 0l.%; and glucose, 1%; pH 7.0;flasks containing the above medium were inoculated with 1% of the sporesuspension described above. The inoculated flasks were incubated for 30hrs. at 28C on a reciprocating shaker set at 65 r.p.m. (4 inch stroke).

Production stage The production stage was run in 250-liter New Brunswickfermenters Model F-250, equipped with automatic antifoam addition systemand pH recordercontroller. The fermenters were charged with liters of anaqueous production medium(8 KM) consisting of the followingconstituents:

soluble starch 1.0% (NHMSO, 0.5% K HOR, 0.5% glucose (Cerelose) 1.5%MgSO, 0.025% ZnSO 0.005% MnSO 0.001% FeSO,.7H,O 0.002% CaCO 0.2%Blackstrap molasses 0.5% hydrolyzed casein (NZ-Case. Sheffield Chemical,Norwich, New York) 0.5% lard oil (Latex No. 1, Swift Canadian Co.,Toronto) 0.2%

pH 7.1 to 7.3

The fermenters were sterilized at 121C for 45 minutes, cooled andinoculated with one flask (2% inoculum) of first stage inoculum.Incubation temperature: 28C; aeration: 0.5 vol/vol/min.; agitation: 2S0r.p.m.

A titre of ca. 20 ug/ml, determined by microbiological assay on agarplates seeded with Candida albicans, was reached in 5 days. Thefermentation was stopped.

Extraction and isolation of the antibiotic was performed by one of thefollowing methods:

Extraction a. The fermentation mixture was extracted twice with 1 v/v ofn-butanol. The combined butanol extracts were washed with 1 v/v ofwater, dried with anhydrous sodium sulfate and evaporated to drynessunder reduced pressure to yield a residue. The oily residue wasextracted 3 times with 2 liters of methanol. The combined methanolextracts were passed through diatomaceous earth (Celite) and evaporatedto dryness to yield an oily residue containing crude rapamyein.

b. The fermentation mixture was filtered over diatomaceous earth(Celite). The filtrate was extracted twice with 1 v/v of ethyl acetate.The ethyl acetate extracts were washed with 1 volume of water, dried 9:1 with anhydrous sodium sulfate and 'evaporated under reduced pressureto dryness. The residuewas extracted twice with 1 liter of methanolfi'lh'e.methanol'extracts aagyroiaryshaker inch stroke) at 240 rpm for 24hrs ISeconid Stage lnoculumnTwenty-four liter flat botwere evaporatedunder reduced pressure to yield-,an

oily residue containing crude rapamycin:

c. The mycelium obtained as'described under section (b) was washed withlto 2 volumes ofwater. The washed mycelium was extracted 3 times with 5volumes of methanol per weight ofwet mycelium ea ch time.

The methanolic extracts were'pooled and concentrated under reducedpressure to a small volume of an aqueous phase containing approximatelyv/v of methanol. This aqueous phase was extracted 3 times withfl vol. ofmethylene chloride; the methylene chlo ride.

extracts were combined, dried withanhydrous sodium sulfate andevaporated to yield an oily residue.

The oily residue was diluted with l volume of petroleum ether, and w/vof charcoal (DarcoiG60) was added. The mixture was stirred for half anhourand filtered. The charcoal, which retainedsubstantially all of theproduct, was washed twice with one volume of petroleum ether. Thecharcoal was elutedthree-times tom flasks containing 3.21 of theinoculum medium described above at pl-l 7.1-7.3 are sterilized-"byautoclaving at 121 for 3 5-"rfiinutes,'shaken to resu spend theinsoluble material "andcresterilized for another 45minutesQThe flasksare icooled to 25 and inoculated with I of the following constituents:

with 5 vol. (based on the weight of the charcoal) of a mixture ofmethylene chloride and ether (50:50). The methylene chloride-etherextracts were evaporated to dryness and the residue dissolved in a smallamount of ether. The crude product was obtained by precipitation fromthe ether solution with cold petroleum ether.

Alternatively, the oily residue obtained by any of the extractionprocedures described above was diluted with 1 vol. of hexane and passedthrough a preparative column of silica gel G. The product was adsorbedon the column. The silica gel G containing adsorbed prod-- uct waswashed with several volumes of hexane and 50:50 hexane-ether mixtures.The product was eluted from the column with ether. The ether eluant wasevaporated to a small volume and crude product obtained by precipitationfrom the ether solution with cold petroleum ether.

Purification EXAMPLE 2 Streptomyces hygroscopicus NRRL 5491 is grown andspores are obtained in the same manner as described in Example 1.

First Stage lnoculum. Erlenmeyer flasks (500 ml) are filled with 100 mlof the following medium:

Soybean flour (Archer-Daniels Co..

Midland, Mich. Special X) 4% wt/vol Glucose (Cerelose) 2% wt/volAmmonium sulfate 0.3% wt/vol Calcium carbonate 0.l5% wt/vol Water tovolume, pH 7.1 to 7.3

The flasks are sterilized at 121 for minutes'and cooled to 25. Theflasks are inoculated with 4% (4 ml) of spore suspension described aboveand incubated on I Soybean flour (Archer-Daniels Co.,

Midland, Mich., Special X") 3% ll wt/vol Glucose (Cerelose) 2% wt/volAmmonium sulfate 0.1% wt/vol Potassium phosphate (monobasic) 0.5% wt/volAntifoaming Agent(DF-1 43 PX? Mazer Chemicals, Inc., Gurnee, lll.,)0.05% wt/vol The-fermenters are sterilizedat 121C for 30 minutes,cooled, and the pH is adjusted to 5.8 to 6.2 with ammonium hydroxide.They are then inoculated with one flask (2%) of second stage inoculumand fermentation is allowed to proceed at 25C, with aeration at 0.25v/v/min and agitation at 200 rpm.

The pH of the fermentation broth starts to drop at 30-35 hours and iscontrolled at 6.0 until the end of fermentation by the automatic, ondemand, addition of ammonium hydroxide. At about 48 hrs. of propagationthe glucose concentration in the broth drops to about 0.5%, andcontinuous addition of 40% glucose solution is started at a rate of3.75% of fermentation mixture volume per day and continued until the endof fermentation. A titer of about 60 ug/ml, determined bymicrobiological assay on agar plates seeded with Candida albicans isreached in 4 to 5 days. The fermentation is stopped at this point.

Extraction and isolation of the antibiotic is performed by the followingprocedure:

The fermentation mixture is filtered over diatomaceous earth (Celite) torecover the mycelium. A typical 400 liter batch obtained from threefermenters yields about 60 kg of wet mycelium. The wet mycelium is mixedwith l vol/wt of methanol by agitation and the mixture is extractedtwice with 2 vol of trichloroethane (methyl chloroform), yielding about250 liters of trichloroethane extract containing about 22-24 g ofrapamycin. The trichloroethane extract is evaporated to dryness underreduced pressure to yield 1 to 1.4 kg of oily residue. This residue isadded slowly with agitation to 5 vols of acetone and the resultingprecipitate is separated by filtration. The acetone solution isevaporated to dryness under reduced pressure to yield an oily residue.This oily residue is extracted twice with 2 and 1 vols of methanolrespectively. The combined methanol extracts are filtered and theremaining oil is discarded. The methanol extract containing rapamycin isevaporated to dryness under reduced pressure to yield 200 to 300 g ofoily residue. This residue is dissolved in 5 v/wt of 15% acetone inhexane. To this solution of the oily residue, silica gel (Merck) isadded in an amount equal to twice the weight of the oily residue and themixture is stirred for about 1 hr. The mixture is filteredon a sinteredglass funnel and the filtrate rejected-.-The silica gel containingrapamycin is washed with several volumes ofl5% acetone in hexane. Thewashed silica gel is eluted with 30 acetone in hexane. The eluant isevaporated to dryness to yield about 60 to 150 g of dry residue. The dryresidue is dissolved in ether and pure rapamycin is separated bycrystallization. A typical run containing about 24 g of crude rapamycinyields between l and 14 g of pure crystalline product.

We claim:

l. Rapamycin, an antibiotic which a. is a colourless, crystallinecompound with a melting point of 183 to l8SC, after recrystallizationfrom ether;

b. is soluble in ether, chloroform, acetone, methanol anddimethylformamide, very sparingly soluble in hexane and petroleum etherand substantially insoluble in water;

c. shows a uniform spot on thin layer plates of silica gel",

d. has a characteristic elemental analysis of about C,

e. exhibits the following characteristic absorption maxima in itsultraviolet absorption spectrum (95% ff has 'a characteristic infraredabsorption spectrum shown in accompanying FIG. 1;

g. has a characteristic nuclear magnetic resonance spectrumlas shown inaccompanying FIG. 2; h. has a minimum inhibitory concentration of 0.02to 0.] pg/ml against Candida albicans; and

i. exhibits a LD (i.p., mice) of 597.3 28.1 mg/kg and a LD (p.o., mice)of 2,500 mg/kg.

2. A process for the production of rapamycin which comprises cultivatingstreptomyces hygroscopicus NRRL 5491 in an aqueous nutrient mediumcontaining a source of assimilable carbon and nitrogen and mineral saltsunder aerobic conditions until substantial antifungal activity ispresent in the fermentation mixture by the production of rapamycin, andisolating rapamycin from said fermentation mixture.

3. A process as claimed in claim 2 in which the cultivation is carriedout'at a temperature ranging from 20 to C and at an initial pH ofbetween 6.5 and 7.5.

4. A process according to claim 2 in which the isolation comprisesfiltering the fermentation mixture, extracting the filter cakewith-methanol or ethanol to produce an extract, and separating therapamycin from the extract. v

1. RAPAMYCIN, AN ANTIBIOTIC WHICH A. IS A LOLOURLESS, CRYSTALLINE COMPOUND WITH A MELTING POINT OF 183* TO 185*C, AFTER RECRYSTALLIZATION FROM ETHER; B. IS SOLUBLE IN ETHER, CHLOROFORM, ACETONE, METHANOL AND DIMETHYLFORMAMIDE, VERY SPARINGLY SOLUBLE IN HEXANE AND PETROLEUM ETHER AND SUBSTANTIALLY INSOLUBLE IN WATER; C. SHOWS A UNIFORM SPOT ON THIN LAYER PLATES OF SILICA GEL; D. HAS A CHARACTERISTIC ELEMENTAL ANALYSIS OF ABOUT C. 66.84%, H, 8.84%; N, 1.37%, E. EXHIBITS THE FOLLOWING CHARACTERISTIC ABSORPTION MAXIMA IN ITS ULTRAVIOLET ABSORPTION SPECTRUM (95% ETHANOL) 267 NM (E1CM1% 417), 277 NM (E1CM1% 541) AND 288 NM (E1CM1% 416); F. HAS A CHARACTERISTIC INFRARED ABSORPTION SPECTRUM SHOWN IN ACCOMPANYING FIG. 1; G. HAS A CHARACTERISTIC NUCLEAR MAGNETIC RESONANCE SPECTRUM AS SHOWN IN ACCOMPANYING FIG. 2; H. HAS A MINIMUM INHIBITORY CONCENTRATION OF 0.002 TO 0.1 UG/ML AGAINST CANDIDA ALBICANS; AND I. EXHIBITS A LD50 (I.P., MICE) OF 597.3 $ 28.1 MG/KG AND A LD50 (P.O., MICE) OF >2,500 MG/KG.
 2. A process for the production of rapamycin which comprises cultivating Streptomyces hygroscopicus NRRL 5491 in an aqueous nutrient medium containing a source of assimilable carbon and nitrogen and mineral salts under aerobic conditions until substantial antifungal activity is present in the fermentation mixture by the production of rapamycin, and isolating rapamycin from said fermentation mixture.
 3. A process as claimed in claim 2 in which the cultivation is carried out at a temperature ranging from 20* to 35*C and at an initial pH of between 6.5 and 7.5.
 4. A process according to claim 2 in which the isolation comprises filtering the fermentation mixture, extracting the filter cake with methanol or ethanol to produce an extract, and separating the rapamycin from the extract. 